RNAi can cause sequence-specific decomposition of mRNA, suppressing expression of a target gene. siRNA and miRNA that cause RNAi hold promise for functional gene analysis and clinical applications because of their technical simplicity and strong gene suppression effects.
In the RNAi pathway, normally double-stranded siRNA or miRNA is incorporated into a protein complex called RISC in the cell, and dessociated into single strands. The strand that remains in the RISC and participates in gene expression is called the guide strand (antisense strand), while the other strand, which is released from the RISC, is called the passenger strand (sense strand). In the case of siRNA, when the guide strand is properly incorporated into the RISC, the RISC complex including the guide strand recognizes a target mRNA having a sequence complementary to the guide strand, and cleaves the target mRNA by agog slicer activity. In the case of miRNA, it recognizes the target mRNA and suppresses the translation process. It is thought that expression of the target gene is suppressed as a result.
It has been hypothesized that of the two strands, RISC distinguishes, as a guide strand, one of the strands from the other one of the strands by a mechanism involving the thermodynamic stability of both ends of the double strand.
In some cases, the passenger strand is incorporated as if the passenger strand were a guide strand. This produces a so-called “off-target” effect. The off-target effect is a phenomenon in which a gene other than the target gene is unintentionally suppressed. In RNAi, when the passenger strand is incorporated into the RISC, the nucleotide sequence of the passenger strand (which is complementary to the guide sequence) may act on the mRNA of a gene other than the original target gene, suppressing its expression.
One method that has been proposed for suppressing this off-target effect is to introduce a modified nucleotide into the passenger strand (Patent Literature 1). It has also been proposed that the passenger strand be provided with a discontinuous site (Patent Literature 2). Another proposal has been to promote incorporation of the antisense strand (guide strand) into the RISC by modifying the 2′ hydroxy groups of the nucleotides of one or both ends of the sense strand (passenger strand) with methyl groups or the like (Patent Literature 3).
It is also known that the PAZ (Piwi/Argonaute/Zwille) domain of the RISC universally has a hydrophobic pocket, and a guide strand-RISC complex is formed by the interaction of the 3′-end of the guide strand with this hydrophobic pocket (Non Patent Literature 1).